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Exploring the Genome and Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical Origin.

ABSTRACT: Klebsiella pneumoniae is an important nosocomial pathogen with an extraordinary resistant phenotype due to a combination of acquired resistant-elements and efflux mechanisms. In this study a detailed molecular characterization of 11 K. pneumoniae isolates of clinical origin was carried out. Eleven clinical isolates were tested for their susceptibilities, by disk diffusion and broth microdilution and interpreted according to CLSI guidelines. Efflux activity was determined by measuring the extrusion of ethidium bromide and biofilm formation was assessed following static growth in Müeller-Hinton and minimal media M9 broths at two temperatures and time points. Template DNA from all 11 isolates was extracted and sequenced. The study collection was found to be resistant to several (extended-spectrum beta-lactam) ESBL-type compounds along with several (fluoro)quinolones (FQ). Resistance to tetracycline accounted for 55% of the study collection (n = 6) and three of the 11 isolates were resistance to carbapenems. Genotyping identified blaCTX-M-15 (82%), blaSHV-12 (55%), and blaTEM-1B (45%) ESBL encoding genes and FQ resistance was associated the presence of the oqxAB operon, identified in 10 of the 11 isolates and qnrB gene in one isolate. The polymorphisms detected in the quinolone resistance-determining regions (QRDRs) were associated with isolates of the clonal group CG15. Sequence types (ST) identified were representative of previously described clonal groups including CG258 (n = 7), CG15 (n = 3), and CG147 (n = 1). Plasmid replicon type databases were queried indicating the presence of IncFII and IncFIB replicon types in the majority of the isolates (91%), followed by IncFIA (45%), and IncR (45%). Two of the 11 isolates were found positive for yersiniabactin siderophore-encoding genes. No differences in the ability to efflux ethidium bromide were identified. Biofilm formation was stronger when the isolates were grown under stressed conditions at 37°C for a period up to 96 h. These data confirm the fact that well-recognized clonal groups of K. pneumoniae of importance to human health carries a diverse repertoire of antimicrobial resistance determinants, particularly related to critically important drugs in the ESBL and FQ classes. The capacity of most isolates to form strong biofilms, when stressed under laboratory-simulated conditions, supports the risk to human health associated with nosocomial infections deriving from indwelling medical devices.

SUBMITTER: Anes J

PROVIDER: S-EPMC5660112 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Exploring the Genome and Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical Origin.

Anes João J Hurley Daniel D Martins Marta M Fanning Séamus S

Frontiers in microbiology 20171023

Klebsiella pneumoniae is an important nosocomial pathogen with an extraordinary resistant phenotype due to a combination of acquired resistant-elements and efflux mechanisms. In this study a detailed molecular characterization of 11 K. pneumoniae isolates of clinical origin was carried out. Eleven clinical isolates were tested for their susceptibilities, by disk diffusion and broth microdilution and interpreted according to CLSI guidelines. Efflux activity was determined by measuri ...[more]

PMID: 29109700

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Project description:BackgroundAntibiotic-resistant Klebsiella pneumoniae has emerged as a critical public health threat worldwide. Understanding the antimicrobial resistance mechanisms of multidrug-resistant K. pneumoniae (MDR-Kp) and its prevalence in time and space would provide clinical significance for managing pathogen infection.MethodsEighteen clinical MDR-Kp strains were analyzed by whole genome sequencing (WGS), and the antimicrobial resistance genes and associated resistance mechanisms were compared with results obtained from the conventional microbiological test (CMT). The sequence homology across strains in our study and those previously collected over time from a wide geographical region was assessed by phylogenetic analysis.ResultsMDR-Kp strains were collected from eighteen patients who had received empirical treatment before strain collection, with sputum (83.3%, 15/18) being the primary source of clinical samples. The commonly received treatments include β-lactamase inhibitors (55.6%, 10/18) and carbapenems (50%, 9/18). Using CMT, we found that all 18 strains were resistant to aztreonam and ciprofloxacin, while 14 (77.8%) showed resistance to carbapenem. Polymyxin B and tigecycline were the only antibiotics to which MDR-Kp strains were sensitive. A total of 42 antimicrobial resistance mechanisms were identified by WGS, surpassing the 40 detected by the conventional method, with 25 mechanisms shared between the two techniques. Despite a 100% accuracy rate of WGS in detecting penicillin-resistant strains, the accuracy in detecting cephalosporin-resistant strains was only at 60%. Among all resistance genes identified by WGS, Klebsiella pneumoniae carbapenemase-2 (KPC-2) was present in all 14 carbapenem-resistant strains. Phenotypic analysis indicated that sequence type (ST) 11 isolates were the primary cause of these MDR-Kp infections. Additionally, phylogenic clustering analysis, encompassing both the clinical and MDR-Kp strains previously reported in China, revealed four distinct subgroups. No significant difference was observed in the sequence homology between K. pneumoniae strains in our study and those previously collected in East China over time.ConclusionThe application of WGS in identifying potential antimicrobial-resistant genes of MDR-Kp has demonstrated promising clinical significance. Comprehensive genomic information revealed by WGS holds the promise of guiding treatment decisions, enabling surveillance, and serving as a crucial asset in understanding antibiotic resistance.

Project description:We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

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Exploring the Genome and Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical Origin.

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Exploring the Genome and Phenotype of Multi-Drug Resistant <i>Klebsiella pneumoniae</i> of Clinical Origin.

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